Project Summary Protein histidine phosphorylation plays an important role in several key signaling pathways in mammals but is very poorly understood. The main reasons for this dearth of information about histidine phosphorylation are the relative instability of pHis as compared to pSer, pThr, and pTyr and a complete lack of chemical tools available to monitor and modulate the enzymes involved in histidine phosphorylation and dephosphorylation. In this project, we will develop fluorogenic assays that can be applied to monitoring both histidine phosphatase and histidine kinase activity and identify the first histidine phosphatase inhibitors from a series of focused screening efforts. This project is submitted in response to PAR-17-046 ?Exploratory Research for Technology Development (R21)? and, as indicated in the PAR, is focused on the development of innovative, enabling technologies for studying histidine phosphorylation and dephosphorylation. Future work would center around applying these tools to developing a better understanding of the roles individual histidine phosphatases in mammalian cells.